In 2006, the worldwide drug market was reported to be worth 643 billion dollars and Korean drug products had an 11,472.8 billion won share of the total. The global drug market is expected to increase to 735˜745 billion dollars in 2007. It was reported that an average cost of 100˜600 million dollars is required to bring a new drug to market, with a developmental period of from 10 to 15 years. In spite of such huge expenses of time and money, the success rate of new drug development is reported to be as low as one in ten thousand. It is accordingly necessary for the successful development of new drugs that highly putative “drug's modes of action” which play critical roles in the occurrence or treatment of diseases, from among a number of possible modes of action, should be defined; this is generally achieved through intelligent bioinformatical tools extracting biological relevance from complex experimental data. Thereby, it is very important to systemically screen specific modes of action directly involved in lots of diseases.
As such, screening technologies are useful in identifying the targets against which drugs exert their therapeutic effects, thus greatly contributing to reducing the time period required for drug development. Also, screening technologies allow the identification of the modes of action which are associated with side effects of the drug. Therefore, there is an imperative need for a novel method for screening a drug's modes of action.
Schizosaccharomyces pombe, a fission yeast useful in the preferable embodiment of the present invention, does not have an evolutionarily high correlation with the budding yeast Saccharomyces cerevisiae although both belong to ascomycetes. S. pombe (Wood V. et al., Nature. 45:871-880, 2002) marked the sixth model eukaryotic organism whose genome has been fully sequenced since S. cerevisiae (Goffeau A. et al., Science, 274:546-567, 1996). According to the analysis, S. pombe has an effective genomic structure which has the lowest functional repetition of genes among the eukaryotic cells whose genomes have been determined thus far. It was also reported that S. pombe contains 4,824 protein-coding genes, which is the smallest number yet identified for a eukaryote, but some 43% of the genes contain introns. Also, S. pombe is found to have highly conserved genes important for eukaryotic cell organization including those required for cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. Further, 31% of the genes of S. pombe were identified to differ from that of S. cerevisiae and to rather share homology with humans. Thus, the comparison of genetic functions between S. pombe and S. cerevisiae is emerging as an effective methodology to study the functions of human genes.
The term “gene targeting”, as used herein to transform yeast strains in the screening of a drug's modes of action, is intended to refer to a genetic technique that uses homologous recombination to change an endogenous gene such as by destroying a gene (knock-out) or by introducing a gene. A transgenic mouse which has had a gene implicated in a specific disease removed therefrom or introduced thereinto has its pathology observed, thus allowing the knock-out gene to be functionally identified.
Since the creation of a transgenic mouse in 1989, gene targeting has made great technical advances. For example, target genes can be introduced at specific developmental stages or into already grown adults. Also, it is possible to design a mutant gene which is expressed at a specific time during development. Martin J. Evans, Oliver Smithies and Mario R. Capecchi were declared laureates of the 2007 Nobel Prize in Physiology and Medicine for their work on gene targeting.
PCR (polymerase chain reaction) is a molecular biological technique for replicating and amplifying DNA (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; European Pat. Nos. 0200362, 0201184 and 0229701; Methods in Enzymology, Volume 155, 1987, pp. 335-350, Murakawa et al., DNA 7; 287-295 (1988)). This technique consists typically of from 30 to 40 cycles of denaturation, annealing and extension, and allows a gene segment of interest to be selectively amplified even from trace amounts in the DNA pools. Now variations on the basic PCR technique have been developed, including multiplex-PCR, nested PCR, quantitative PCR, real-time PCR, reverse transcription (RT-PCR), touchdown PCR, etc.
Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification, based on the detection of a fluorescent signal produced proportionally during amplification of a PCR product, because a fluorescent probe is used together with primers. Real-time PCR can obtain PCR results rapidly and conveniently because it does not need electrophoretic determination. Also, real-time PCR enjoys the advantage of a low risk of contamination. For these reasons, real-time PCR enjoys widespread use as a substitute for typical PCR.
Transformation is the genetic alteration of a cell resulting from the uptake, genomic incorporation, and expression of foreign DNA. In practice, the foreign DNA is labeled with a selectable marker to easily select the cells to which the foreign DNA has been successfully introduced. Typical examples of the selectable markers include ampicillin-, tetracycline- and kanamycin-resistance genes. After transformation, culturing in the presence of related antibiotics allows the selection of successfully transformed bacteria. In the present invention, a specific gene is knocked out in the fission yeast by transformation with the introduction of an antibiotic-resistance gene into the corresponding homologous recombination site.
Gene synthesis is a well-known technique on which about 200 articles have published since the development of oligo-ligation based on PCR in the early 1990s (Edge et al., Nature. 292:756-62, Rouillard et al., NAR. 32:176-80, Smith et al., NAR. 10:4467-82, Dillon et al., Biotechniques. 9:298-300, Ciccarelli et al., Nucleic Acid Research. 21:6007-13, Prodromou et al., Protein Engineering. 5:827-29, Stemmer et al., Gene. 164:49-53, Lin et al., Gene. 288:85-94, Venter et al., Proceedings National Academy of Science. 100:15440-45). In principle, oligonucleotides from 20 bp to 60 bp long are annealed and ligated to each other to give a double-stranded DNA fragment which is then used as a template for the amplification of a desired DNA through PCR. The DNA fragment which can be obtained by gene synthesis is generally shorter than 1,000 bp. For a longer gene, the obtained DNA fragments less than 1000 bp are ligated by PCR. In 2003, Dr. Craig Venter succeeded in building the entire 5,386-bp genome of ΦX174 bacteriophage (phiX174 bacteriophage) in such a manner.
With regard to the screening of a drug's mode of action, most widely used are in vitro methods. For example, a mixture of cellular proteins are passed through an affinity column with a drug attached on a resin, and the proteins thus bound to the drug are purified and analyzed by MALDI-TOF (Schreiber et al., Bioorg. Med. Chem. 6:1127-1152). An alternative is to pass a human protein-phage library in which human proteins are expressed on the membrane through an affinity column containing drug-attached resin, followed by selection and analysis of phages bound to the drug to infer the target proteins of the drug (Sche et al., Chem. Biol. 6:707-716).
Recently, attempts have been made on an in vivo screening method which is quite different from the above two methods (Lum et al Cell. 116:121-37). This method, although disadvantageous in that it screens yeast proteins in contrast to the in vitro methods of screening human proteins, has the advantage of being rapid (100 drugs/week), convenient and having a high success rate. In 2004, Drs. Shoemaker and Lum's research team at Merck screened modes of action against 80 pre-existing drugs with an increase in success rate of from 25% to 70%. The screening strategy, referred to as drug-induced haploinsufficiency, is based on the fact that lowering the gene dosage of a drug target increases the susceptibility to the drug. In most cases, one allele is sufficient to permit the cell to function normally, but when the normal phenotype requires the protein product of both alleles, and restitution of 50% of the gene function results in an abnormal phenotype, this is referred to as haploinsufficiency. It was reported that 180 genes, corresponding to 3% of about 6,000 genes, of budding yeast, were implicated in haploinsufficiency such that the mutants grew poorly even in sufficient nutrient media compared to the wild-type (Deutschbauer et al., Genetics. 169:1915-25). This haploinsufficiency is a basis for screening a drug's modes of action in heterozygous mutant yeast strains. For example, assuming that a drug ‘a’ targets a protein ‘A’, treatment with the drug ‘a’ at a dose of IC50, a concentration to inhibit the protein ‘A’ by half; leads to 100% inhibition (knock-down) of the protein ‘A’ in the gene-targeted heterozygous strain. Accordingly, the drug-treated strain grows at a lower rate than does the non-treated strain. In accordance with the present invention, a method for screening a drug's modes of action on the basis of haploinsufficiency is provided.
Success in constructing gene targeted strains is dependent on the properties of the strains because it is determined by homologous recombination efficiency which differs from one strain to another. However, the homologous recombination efficiency in the same strain is proportional to the length of genomic homologous sites. Thus, a longer DNA fragment corresponding to a genomic homologous site permits the construction of a gene-targeted strain at a more efficient rate. The trials summarized in Table 1 below have been performed on budding yeast, but there are no precedents on fission yeast at such a high success rate (Palaniyandi et al., Nucl Acid Res. 3:2799-2800; Michael et al., Gene. 158:113-17; Kaur et al., Nucl Acid Res. 25:1080-81).
TABLE 1HomologousSuccessConstruction of Deletion CassettesYeastsLength (bp)rate(%)PCR with gene-Serial PCRBudding45~6081specific OligoSingle OligoBudding38~5010Single OligoFission401~3
Typically, a gene specific linker-mediated PCR method has been used to lengthen homologous recombination sites for use in the construction of gene-targeted fission yeast strains. Using this method, Giaever et al. constructed about 6000 deletion cassettes of the total genome of the budding yeast such that each cassette was flanked by two 40-60 bp homologous regions of yeast DNA sequence (Giaever et al., Nature Genetics 14:450-56; Wach et al., Yeast 10:1793-1808). In the case of the budding yeast, 45˜60 bp genomic homologous regions at both termini of each cassette guaranteed a success rate of as high as 81% in the genome-wide construction of mutant strains (Table 1). In fission yeast however, even when the homologous length is extended to 40 bp, the success rate is increased to at most 1˜3%. Such a low success rate for the fission yeast indicates that strain preparation is very difficult due to the inferiority of the fission yeast in homologous recombination rate to that of the budding yeast. In full consideration of this background, the present invention provides a method for constructing fission yeast mutants at high efficiency through homologous recombination.